Adjustable bilayer capacitance structure for biomedical devices

ABSTRACT

A nanopore sequencing device is disclosed. The nanopore sequencing device includes a working electrode. It further includes a dielectric layer, wherein a portion of the dielectric layer is disposed horizontally adjacent to the working electrode and a portion of the dielectric layer is disposed above and covering a portion of the working electrode, and wherein the dielectric layer forms a well having an opening above an uncovered portion of the working electrode. A base surface area of the working electrode is greater than a base surface area of the opening above the uncovered portion of the working electrode.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/021,664, entitled ADJUSTABLE BILAYER CAPACITANCE STRUCTURE FORBIOMEDICAL DEVICES, filed Jun. 28, 2018, which is a continuation of U.S.patent application Ser. No. 14/606,632 entitled ADJUSTABLE BILAYERCAPACITANCE STRUCTURE FOR BIOMEDICAL DEVICES filed Jan. 27, 2015, nowU.S. Pat. No. 10,036,739, each of which is incorporated herein byreference for all purposes.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state.

FIG. 7A illustrates an additional embodiment of a circuitry 700 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7B illustrates an additional embodiment of a circuitry 701 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane.

FIG. 9 illustrates an embodiment of a plot of the voltage applied acrossthe nanopore versus time when process 800 is performed and repeatedthree times.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates.

FIG. 11 illustrates an embodiment of a cell 1100 in a nanopore basedsequencing chip.

FIG. 12 illustrates an embodiment of a cell 1200 in a nanopore basedsequencing chip.

FIG. 13 illustrates an embodiment of a process for constructing a cellin a nanopore based sequencing chip, wherein the capacitancesC_(membrane) and C_(dl) in the cell may be adjusted independently byadjusting the base surface area of the membrane and the base surfacearea of the working electrode separately.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly ontothe surface of the cell. A single PNTMC 104 is inserted into membrane102 by electroporation. The individual membranes in the array areneither chemically nor electrically connected to each other. Thus, eachcell in the array is an independent sequencing machine, producing dataunique to the single polymer molecule associated with the PNTMC. PNTMC104 operates on the analytes and modulates the ionic current through theotherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a thin film of electrolyte108. The thin film of electrolyte 108 is isolated from the bulkelectrolyte 114 by the ion-impermeable membrane 102. PNTMC 104 crossesmembrane 102 and provides the only path for ionic current to flow fromthe bulk liquid to working electrode 110. The cell also includes acounter electrode (CE) 116, which is an electrochemical potentialsensor. The cell also includes a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. At stage A, a tagged nucleotide (one offour different types: A, T, G, or C) is not associated with thepolymerase. At stage B, a tagged nucleotide is associated with thepolymerase. At stage C, the polymerase is in close proximity to thenanopore. The tag is pulled into the nanopore by an electrical fieldgenerated by a voltage applied across the membrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage B.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 pico Siemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS corresponding toone of the four types of tagged nucleotides. The polymerase undergoes anisomerization and a transphosphorylation reaction to incorporate thenucleotide into the growing nucleic acid molecule and release the tagmolecule. In particular, as the tag is held in the nanopore, a uniqueconductance signal (e.g., see signal 210 in FIG. 2) is generated due tothe tag's distinct chemical structures, thereby identifying the addedbase electronically. Repeating the cycle (i.e., stage A through E orstage A through F) allows for the sequencing of the nucleic acidmolecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip. As mentioned above, when the tag is heldin nanopore 502, a unique conductance signal (e.g., see signal 210 inFIG. 2) is generated due to the tag's distinct chemical structures,thereby identifying the added base electronically. The circuitry in FIG.5 maintains a constant voltage across nanopore 502 when the current flowis measured. In particular, the circuitry includes an operationalamplifier 504 and a pass device 506 that maintain a constant voltageequal to V_(a) or V_(b) across nanopore 502. The current flowing throughnanopore 502 is integrated at a capacitor n_(cap) 508 and measured by anAnalog-to-Digital (ADC) converter 510.

However, circuitry 500 has a number of drawbacks. One of the drawbacksis that circuitry 500 only measures unidirectional current flow. Anotherdrawback is that operational amplifier 504 in circuitry 500 mayintroduce a number of performance issues. For example, the offsetvoltage and the temperature drift of operational amplifier 504 may causethe actual voltage applied across nanopore 502 to vary across differentcells. The actual voltage applied across nanopore 502 may drift by tensof millivolts above or below the desired value, thereby causingsignificant measurement inaccuracies. In addition, the operationalamplifier noise may cause additional detection errors. Another drawbackis that the portions of the circuitry for maintaining a constant voltageacross the nanopore while current flow measurements are made arearea-intensive. For example, operational amplifier 504 occupiessignificantly more space in a cell than other components. As thenanopore based sequencing chip is scaled to include more and more cells,the area occupied by the operational amplifiers may increase to anunattainable size. Unfortunately, shrinking the operational amplifier'ssize in a nanopore based sequencing chip with a large-sized array mayraise other performance issues. For example, it may exacerbate theoffset and noise problems in the cells even further.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. One of the possible statesof the nanopore is an open-channel state when a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Another fourpossible states of the nanopore correspond to the states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. FIGS. 7A and 7B illustrateadditional embodiments of a circuitry (700 and 701) in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. In the above circuits, theoperational amplifier is no longer required.

FIG. 6 shows a nanopore 602 that is inserted into a membrane 612, andnanopore 602 and membrane 612 are situated between a cell workingelectrode 614 and a counter electrode 616, such that a voltage isapplied across nanopore 602. Nanopore 602 is also in contact with a bulkliquid/electrolyte 618. Note that nanopore 602 and membrane 612 aredrawn upside down as compared to the nanopore and membrane in FIG. 1.Hereinafter, a cell is meant to include at least a membrane, a nanopore,a working cell electrode, and the associated circuitry. In someembodiments, the counter electrode is shared between a plurality ofcells, and is therefore also referred to as a common electrode. Thecommon electrode can be configured to apply a common potential to thebulk liquid in contact with the nanopores in the measurements cells. Thecommon potential and the common electrode are common to all of themeasurement cells. There is a working cell electrode within eachmeasurement cell; in contrast to the common electrode, working cellelectrode 614 is configurable to apply a distinct potential that isindependent from the working cell electrodes in other measurement cells.

In FIGS. 7A and 7B, instead of showing a nanopore inserted in a membraneand the liquid surrounding the nanopore, an electrical model 702representing the electrical properties of the nanopore and the membraneand an electrical model 714 representing the electrical properties ofthe working electrode are shown. Electrical model 702 includes acapacitor 706 that models a capacitance associated with the membrane(C_(membrane)) and a resistor 704 that models a resistance associatedwith the nanopore in different states (e.g., the open-channel state orthe states corresponding to having different types of tag/moleculeinside the nanopore). Electrical model 714 includes a capacitor 716 thatmodels a capacitance associated with the working electrode. Thecapacitance associated with the working electrode is also referred to asa double layer capacitance (C_(dl)). Note in FIGS. 7A and 7B that therespective circuitry does not require an extra capacitor (e.g., n_(cap)508 in FIG. 5) that is fabricated on-chip, thereby facilitating thereduction in size of the nanopore based sequencing chip.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane. Process 800 may be performed using the circuitries shown inFIG. 6, 7A, or 7B. FIG. 9 illustrates an embodiment of a plot of thevoltage applied across the nanopore versus time when process 800 isperformed and repeated three times. The voltage across the nanoporechanges over time. The rate of the voltage decay (i.e., the steepness ofthe slope of the voltage across the nanopore versus time plot) dependson the cell resistance (e.g., the resistance of resistor 704 in FIG.7A). More particularly, as the resistances associated with the nanoporein different states (e.g., the open-channel state, the statescorresponding to having different types of molecules inside the nanoporeare different due to the molecules' distinct chemical structure,different corresponding rates of voltage decay may be observed and thusmay be used to identify the molecule in the nanopore.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates. Curve 1002 shows the rate of voltage decay during anopen-channel state. In some embodiments, the resistance associated withthe nanopore in an open-channel state is in the range of 100 Mohm to 20Gohm. Curves 1004, 1006, 1008, and 1010 show the different rates ofvoltage decay corresponding to the four capture states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. In some embodiments, the resistanceassociated with the nanopore in a capture state is within the range of200 Mohm to 40 Gohm. Note that the slope of each of the plots isdistinguishable from each other.

Allowing the voltage applied across the nanopore to decay over a timeperiod during which the nanopore is in a particular detectable state hasmany advantages. One of the advantages is that the elimination of theoperational amplifier, the pass device, and the capacitor (e.g., n_(cap)508 in FIG. 5) that are otherwise fabricated on-chip in the cellcircuitry significantly reduces the footprint of a single cell in thenanopore based sequencing chip, thereby facilitating the scaling of thenanopore based sequencing chip to include more and more cells (e.g.,having millions of cells in a nanopore based sequencing chip). Thecapacitance in parallel with the nanopore includes two portions: thecapacitance associated with the membrane and the capacitance associatedwith the integrated chip (IC). Due to the thin nature of the membrane,the capacitance associated with the membrane alone can suffice to createthe required RC time constant without the need for additional on-chipcapacitance, thereby allowing significant reduction in cell size andchip size.

Another advantage is that the circuitry of a cell does not suffer fromoffset inaccuracies because V_(pre) is applied directly to the workingelectrode without any intervening circuitry. Another advantage is thatsince no switches are being opened or closed during the measurementintervals, the amount of charge injection is minimized.

Furthermore, the technique described above operates equally well usingpositive voltages or negative voltages. Bidirectional measurements havebeen shown to be helpful in characterizing a molecular complex. Forexample, they can be used to correct for baseline drift arising fromAC-non-faradaic operation.

The ratio of the capacitance associated with the membrane (seeC_(membrane) 706 of FIGS. 7A and 7B) and the capacitance associated withthe working electrode (see C_(dl) 716 of FIGS. 7A and 7B) may beadjusted to achieve optimal overall system performance. For example,increased system performance may be achieved by reducing C_(membrane)while maximizing C_(dl). In another example, C_(membrane) is adjusted tocreate the required RC time constant without the need for additionalon-chip capacitance, thereby allowing a significant reduction in cellsize and chip size. In another example, C_(dl) is maximized such thatthe impedance associated with C_(dl) is close to an AC (alternatingcurrent) short circuit compared with the impedance associated withC_(membrane).

FIG. 11 illustrates an embodiment of a cell 1100 in a nanopore basedsequencing chip. In this embodiment, the ratio of the C_(membrane) andC_(dl) may be adjusted by increasing C_(dl), as will be described ingreater detail below.

Cell 1100 includes a dielectric layer 1101. Dielectric material used toform dielectric layer 1101 includes glass, oxides, nitrides, and thelike. Cell 1100 further includes a dielectric layer 1104 abovedielectric layer 1101. Dielectric layer 1104 forms the walls surroundinga well 1105 in which a working electrode 1102 is located at the bottom.Dielectric material used to form dielectric layer 1104 includes glass,oxide, silicon mononitride (SiN), and the like. The top surface ofdielectric layer 1104 may be silanized. Silanization forms a hydrophobiclayer 1120 above the top surface of dielectric layer 1104. In someembodiments, hydrophobic layer 1120 has a thickness of about 1.5nanometer (nm).

Well 1105 formed by the dielectric layer walls 1104 further includes afilm of salt solution 1106 above working electrode 1102. Salt solution1106 may include one of the following: lithium chloride (LiCl), sodiumchloride (NaCl), potassium chloride (KCl), lithium glutamate, sodiumglutamate, potassium glutamate, lithium acetate, sodium acetate,potassium acetate, calcium chloride (CaCl₂), strontium chloride (SrCl₂),Manganese chloride (MnCl₂), and magnesium chloride (MgCl₂). In someembodiments, the film of salt solution 1106 has a thickness of aboutthree microns (μm).

As shown in FIG. 11, a membrane is formed on top of dielectric layer1104 and spans across well 1105. For example, the membrane includes alipid monolayer 1118 formed on top of hydrophobic layer 1120. As themembrane reaches the opening of well 1105, the lipid monolayertransitions to a lipid bilayer 1114 that spans across the opening of thewell. A bulk electrolyte 1108 containing protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly above the well. A single PNTMC/nanopore 1116 is inserted intolipid bilayer 1114 by electroporation. Nanopore 1116 crosses lipidbilayer 1114 and provides the only path for ionic flow from bulkelectrolyte 1108 to working electrode 1102. Bulk electrolyte 1108 mayfurther include one of the following: lithium chloride (LiCl), sodiumchloride (NaCl), potassium chloride (KCl), lithium glutamate, sodiumglutamate, potassium glutamate, lithium acetate, sodium acetate,potassium acetate, calcium chloride (CaCl₂), strontium chloride (SrCl₂),Manganese chloride (MnCl₂), and magnesium chloride (MgCl₂).

Cell 1100 includes a counter electrode (CE) 1110, which is anelectrochemical potential sensor. Cell 1100 also includes a referenceelectrode 1112. In some embodiments, counter electrode 1110 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 1102 is a metal electrode. Fornon-faradaic conduction, working electrode 1102 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 1102 maybe a platinum electrode with electroplated platinum.

As discussed above, the ratio of C_(membrane) and C_(dl) in cell 1100may be adjusted by increasing C_(dl). The double layer capacitance(C_(dl)) associated with working electrode 1102 may be increased byincreasing the thickness of working electrode 1102. In some embodiments,the thickness of working electrode 1102 ranges from 10 nanometers to 1micron.

C_(dl) may also be increased by maximizing the surface area of workingelectrode 1102 for a given volume. As the surface area increases, thecapacitance of the double layer (C_(dl)) increases, and a greater amountof ions can be displaced with the same applied potential before thecapacitor becomes charged. For example, the surface area of the workingelectrode may be increased by making the electrode “spongy.” In someembodiments, the capacitance of the double layer can be enhanced byelectroplating platinum metal onto a 5 micron diameter smooth platinumelectrode in the presence of a detergent. The detergent createsnanoscale interstitial spaces in the platinum metal, making it “spongy.”The platinum sponge soaks up electrolyte and creates a large effectivesurface area (e.g., 33 pF per square micron of electrode top-down area).

Another way to increase C_(dl) is by increasing the base surface area ofworking electrode 1102. For example, if the working electrode has acylindrical shape, then the base surface area of the cylinder may beincreased. In another example, if the working electrode has arectangular prism shape, then the base surface area of the rectangularprism may be increased. However, cell 1100 has a drawback. Workingelectrode 1102 and lipid bilayer 1114 have the same (or similar) basesurface area or cross sectional area. When the base surface area ofworking electrode 1102 is increased, the base surface area of theopening of well 1105 and lipid bilayer 1114 are both increased as well.As a result, both C_(membrane) and C_(dl) are increased simultaneously.In other words, to optimize the overall system performance, C_(membrane)cannot be reduced while maximizing C_(dl) by adjusting the base area ofwell 1105 alone.

FIG. 12 illustrates an embodiment of a cell 1200 in a nanopore basedsequencing chip. In contrast to cell 1100, C_(membrane) and C_(dl) incell 1200 may be adjusted independently by adjusting the base surfacearea of the membrane and the base surface area of the working electrodeseparately.

Cell 1200 includes a dielectric layer 1201. Cell 1200 further includes aworking electrode 1202 and a dielectric layer 1203 above dielectriclayer 1201. In some embodiments, working electrode 1202 is circular inshape and dielectric layer 1203 forms the walls surrounding workingelectrode 1202. Cell 1200 further includes a dielectric layer 1204 aboveworking electrode 1202 and dielectric layer 1203. Dielectric layer 1204forms the walls surrounding a well 1205. In some embodiments, dielectriclayer 1203 and dielectric layer 1204 together form a single piece ofdielectric. Dielectric layer 1203 is the portion that is disposedhorizontally adjacent to working electrode 1202, and dielectric layer1204 is the portion that is disposed above and covering a portion of theworking electrode. The dielectric forms well 1205, which has an openingabove an uncovered portion of the working electrode. In someembodiments, dielectric layer 1203 and dielectric layer 1204 areseparate pieces of dielectric and they may be grown separately.

Inside well 1205, a film of salt solution 1206 is deposited aboveworking electrode 1202. Salt solution 1206 may include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂). In some embodiments, the filmof salt solution 1206 has a thickness of about three microns. Thethickness of the film of salt solution 1206 may range from 0-5 microns.

Dielectric material used to form dielectric layers 1201, 1203, and 1204includes glass, oxide, silicon mononitride (SiN), and the like. The topsurface of dielectric layer 1204 may be silanized. Silanization forms ahydrophobic layer 1220 above the top surface of dielectric layer 1204.In some embodiments, hydrophobic layer 1220 has a thickness of about 1.5nanometer (nm).

As shown in FIG. 12, a membrane is formed on top of dielectric layer1204 and spans across well 1205. For example, the membrane includes alipid monolayer 1218 formed on top of hydrophobic layer 1220 and as themembrane reaches the opening of well 1205, the lipid monolayertransitions to a lipid bilayer 1214 that spans across the opening of thewell. Hydrophobic layer 1220 facilitates the formation of lipidmonolayer 1218 above dielectric layer 1204 and the transition from alipid monolayer to a lipid bilayer. A bulk electrolyte 1208 containingprotein nanopore transmembrane molecular complexes (PNTMC) and theanalyte of interest is placed directly above the well. A singlePNTMC/nanopore 1216 is inserted into lipid bilayer 1214 byelectroporation. Nanopore 1216 crosses lipid bilayer 1214 and providesthe only path for ionic flow from bulk electrolyte 1208 to workingelectrode 1202. Bulk electrolyte 1208 may further include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂).

In cell 1200, the base surface area of the opening of well 1205 (whichis the same as the base surface area of lipid bilayer 1214) and the basesurface area of working electrode 1202 are determined by the dimensionsof dielectric layer 1204 and dielectric layer 1203, respectively. Thebase surface area of working electrode 1202 is greater than or equal tothe base surface area of the opening of well 1205.

Cell 1200 includes a counter electrode (CE) 1210, which is anelectrochemical potential sensor. Cell 1200 also includes a referenceelectrode 1212. In some embodiments, counter electrode 1210 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 1202 is a metal electrode. Fornon-faradaic conduction, working electrode 1202 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 1202 maybe a platinum electrode with electroplated platinum.

Similar to cell 1100, the ratio of C_(membrane) and C_(dl) in cell 1200may be adjusted by increasing C_(dl). The double layer capacitance(C_(dl)) associated with working electrode 1202 may be increased byincreasing the thickness of working electrode 1202. In some embodiments,the thickness of working electrode 1202 ranges from 10 nanometers to 1micron.

C_(dl) may also be increased by maximizing the surface area of workingelectrode 1202 for a given volume. For example, the surface area of theworking electrode may be increased by making the electrode “spongy.” Insome embodiments, the capacitance of the double layer can be enhanced byelectroplating platinum metal onto a 5 micron diameter smooth platinumelectrode in the presence of a detergent.

Another way to adjust the ratio of C_(membrane) and C_(dl) is byadjusting the base surface area of the opening of well 1205 and the basesurface area of working electrode 1202 independently from each other. Incell 1200, the base surface area of the opening of well 1205 (which isthe same as the base surface area of lipid bilayer 1214) and the basesurface area of working electrode 1202 are determined by the dimensionsof dielectric layer 1204 and dielectric layer 1203, respectively.Therefore, the two base surface areas may be optimized independently toprovide the desired ratio between C_(membrane) and C_(dl). For example,as shown in FIG. 12, the base surface area of working electrode 1202 iskept at the same size as working electrode 1102 in FIG. 11, while thebase surface area of the opening of well 1205 is reduced, therebyreducing C_(membrane), while maximizing C_(dl).

In some embodiments, the diameters of working electrode 1202 and theopening of well 1205 range from 0.5 to 6 microns. C_(membrane) has acapacitance that ranges from 5 to 300 femto farad (fF).

FIG. 13 illustrates an embodiment of a process for constructing a cellin a nanopore based sequencing chip, wherein C_(membrane) and C_(dl) inthe cell may be adjusted independently by adjusting the base surfacearea of the membrane and the base surface area of the working electrodeseparately. At step A, a layer of dielectric 1 is disposed on top of ametal 6 layer (M6). In some embodiments, the layer of dielectric 1 has athickness of about 400 nm. At step B, the layer of dielectric 1 isetched to create a well. At step C, a layer of metal or metal oxide isdeposited to fill the well created at step B. At step D, the excessmetal or metal oxide is removed. For example, the excess metal or metaloxide may be removed using chemical mechanical polishing (CMP)techniques. The remaining metal or metal oxide deposited in the wellforms a working electrode. After the working electrode is formed, alayer of dielectric 2 is deposited on top of the dielectric 1 and theworking electrode. At step E, the layer of dielectric 2 is etched tocreate a well exposing only a portion of the upper base surface surfacearea of the working electrode. Because the base surface area of theopening of the well is independent from the base surface area of theworking electrode, C_(membrane) and C_(dl) in the cell may be fine tunedto obtain the desired C_(membrane) and C_(dl) ratio.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A nanopore sequencing device, the devicecomprising: a working electrode having a first diameter or width; and adielectric layer disposed over the working electrode; and a well formedin the dielectric layer over the working electrode, the well having asidewall formed from the dielectric layer and an opening above theworking electrode, the opening of the well having a second diameter orwidth; wherein the first diameter or width of the working electrode isgreater than the second diameter or width of the opening of the well. 2.The device of claim 1, wherein the working electrode is a non-faradaicelectrode.
 3. The device of claim 1, wherein the working electrode ismade of titanium nitride.
 4. The device of claim 1, wherein the workingelectrode is disposed on top of a metal layer.
 5. The device of claim 1,wherein the first diameter and the second diameter are between 0.6microns to 6 microns.
 6. The device of claim 1, wherein the well isconfigured to support a membrane that extends across the opening of thewell.
 7. A nanopore sequencing device, the device comprising: an arrayof wells formed in a layer of dielectric material, each well having: asidewall formed from the layer of dielectric material, an opening at atop of the well, and a working electrode at a bottom of the well,wherein the working electrode has a diameter greater than a diameter ofthe opening at the top of the well.
 8. The device of claim 7, whereinthe layer of dielectric material and the working electrode of each wellin the array are disposed on top of a metal layer.
 9. The device ofclaim 7, wherein each well is configured to support a membrane thatextends across the opening of the well.
 10. The device of claim 7,wherein both the diameter of the working electrode and the diameter ofthe opening are between 0.6 microns to 6 microns.
 11. The device ofclaim 7, wherein the working electrode is a non-faradaic electrode. 12.The device of claim 7, wherein the working electrode is made of titaniumnitride.
 13. A method of fabricating a sequencing device, the methodcomprising: disposing a first dielectric layer over a substrate; forminga cavity in the first dielectric layer having a first diameter or width;disposing a working electrode material in the cavity to form a workingelectrode having a first diameter or width; disposing a seconddielectric layer over both the first dielectric layer and the workingelectrode; forming a well in the second dielectric layer over theworking electrode, the well having an opening with a second diameter orwidth and a base formed by the working electrode, wherein the firstdiameter or width is greater than the second diameter or width.
 14. Themethod of claim 13, wherein the step of disposing the working electrodematerial in the cavity further comprises disposing the working electrodematerial over the first dielectric layer.
 15. The method of claim 13,further comprising planarizing the working electrode material and thefirst dielectric layer after the step of disposing the working electrodematerial in the cavity and over the first dielectric layer.
 16. Themethod of claim 14, wherein the working electrode is planarized using achemical mechanical polishing technique.
 17. The method of claim 13,wherein the first dielectric layer is disposed on a metal layer.
 18. Themethod of claim 13, wherein the first diameter and the second diameterare between 0.6 microns to 6 microns.